CDNA encoding the type I iodothyronine 5'deiodinase

• US 5,272,078 A
• Filed: 01/29/1992
• Issued: 12/21/1993
• Est. Priority Date: 01/29/1991
• Status: Expired due to Fees

• Current Assignee: Brigham and Women's Hospital Incorporated
• Original Assignee: Brigham and Women's Hospital Incorporated
• Inventors: Larsen, P. Reed, Berry, Marla J.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US07/828,790
• Time in Patent Office: 692 Days
• Field of Search: 435/69.1, 435/252.3, 435/320.1, 435/187, 536/27, 536/23.2, 536/24.1
• US Class Current: 435/189
• CPC Class Codes: C07K 14/61 Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 9/0065 acting on hydrogen peroxide as acceptor (1.11)

Abstract

The present invention is directed to the cloning, sequencing, and uses of a nucleotide sequence encoding the mammalian enzyme Type I iodothyronine 5'"'"' deiodinase and mutant sequences thereof. This enzyme catalyzes the conversion of thyroxine to triiodothyronine, producing the active form of thyroid hormone. The invention is further directed to the discovery that an untranslated nucleotide sequence is necessary for the recognition of a TGA (termination) codon in the coding region of 5'"'"' deiodinase as encoding selenocysteine, an amino acid essential for the full activity of 5'"'"' deiodinase. The mechanism by which this recognition occurs involves a stem-loop structure in the 3'"'"' untranslated region of the mRNA. The untranslated sequence can be used to incorporate selenocysteine into non-selenocysteine-containing proteins and polypeptides.

Claims

1. An isolated DNA molecule consisting essentially of a DNA segment encoding an enzyme having the activity of Type I iodothyronine 5'"'"'-deiodinase.

2. The isolated DNA molecule of claim 1 wherein said enzyme is encoded by the sequence as shown in FIG. 4B (and Sequence ID5), or a sequence encoding a functional derivative thereof.

8. A linear vector comprising the isolated DNA molecule of claim 1.

9. The linear vector of claim 8, further comprising a DNA sequence which directs expression of said enzyme.

10. The linear of vector of claim 8 further comprising a DNA sequence encoding a selenocysteine-containing polypeptide operably linked to said DNA molecule and a second DNA sequence capable of directing expression of said polypeptide.

11. A host cell transformed with the linear vector of claim 8, said host cell being capable of expressing an enzyme having the activity of Type I iodothyronine 5'"'"' deiodinase.

3. A method of producing a polypeptide containing at least one selenocysteine residue at a desired site, said method comprising:

(a) preparing an expression vector comprising

(i) a first DNA sequence encoding said polypeptide, wherein said first DNA sequence contains a TGA codon at a site corresponding to the desired selenocysteine site in said polypeptide;

(ii) a promoter region capable of directing transcription of said first DNA sequence wherein said promoter region is operably linked to the 5'"'"' end of said first DNA sequence; and

(iii) a second DNA sequence wherein said second DNA sequence encodes a selenocysteine-insertion sequence and wherein said second sequence is operably linked 3'"'"' to said first DNA sequence and wherein said second DNA sequence is capable of directing the incorporation of selenocysteine into said polypeptide;

(b) transforming a host cell culture with said expression vector to obtain a transformed, recombinant host cell;

(c) culturing said recombinant host cell under conditions permitting expression and of said protein; and

(d) recovering said protein.

4. The method of claim 3 wherein said selenocysteine-insertion sequence is encoded by nucleotides 1573-1894 as shown in FIG. 4B (and Sequence ID5), or a functional derivative thereof.

5. An isolated reporter gene consisting essentially of a DNA sequence encoding a functional Type I 5'"'"' deiodinase.

6. The reporter gene of claim 5 wherein the sequence encoding Type I 5'"'"'-deiodinase is that shown in FIG. 4B (and Sequence ID 5).

7. The reporter gene of claim 5 wherein the codon for selenocysteine is replaced with that for cysteine.

12. An isolated selenocysteine-insertion sequence which, when operably linked 3'"'"' to a DNA sequence encoding a selenocysteine-containing polypeptide, effects incorporation of selenocysteine into said polypeptide.

13. The isolated selenocysteine-insertion sequence of claim 12 which is encoded by nucleotides 1573-1894 as shown in FIG. 4B (and Sequence ID 5).

14. An expression vector for the synthesis of a selenocysteine-containing polypeptide, wherein said expression vector comprises:

(a) a first DNA sequence encoding said selenocysteine-containing polypeptide operably linked to a promoter region; and

(b) a second DNA sequence which encodes a selenocysteine-insertion sequence, wherein said second DNA sequence is operably linked to said first DNA sequence and said second DNA sequence directs the incorporation of selenocysteine into said selenocysteine-containing polypeptide, and wherein said second DNA sequence is not translated.

15. A host cell transformed with the expression vector of claim 14.